MS/MS Data Processing

ABSTRACT

A method of identifying precursor ion species from their fragments comprises obtaining mass spectra of a plurality of precursor ion species and their fragments to high mass accuracy. The fragment mass spectrum, obtained from fragmentation of multiple precursor ion species, is then scanned it identify pairs of fragments whose combined mass matches the mass of one of the precursor ion species. Once pairs of fragment ion shave been matched to precursor ions, the composite fragment ion spectrum is broken down into portions, one per fragment pair. Analysis continues until no further pairs are identified. A simplified fragment ion spectrum is then reconstructed for each precursor sample ion by stitching together the broken down sections of the composite fragment spectrum. The resultant reconstructed, simplified fragment spectra are sent to a search engine which returns a score—sorted list of likely candidates for each synthetic fragment ion spectrum.

CROSSREFERENCE TO RELATED APPLICATIONS

The present application is a continuation under 35 U.S.C. §120 andclaims the priority benefit of co-pending U.S. patent application Ser.No. 12/992,839, filed Jan. 14, 2011, which is a National Stageapplication under 35 U.S.C. §371 of PCT Application No.PCT/EP2009/003175, filed May 4, 2009. The disclosures of each of theforegoing applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of mass spectrometry andmore specifically to a method of identifying precursor ion species fromtheir fragments (MS/MS data processing).

BACKGROUND OF THE INVENTION

The mass spectrometric analysis of molecules is complicated by thepresence of many different molecules having closely similar mass tocharge ratios. Fragmentation techniques have been developed to helpidentify the different parent molecules by measuring the mass to chargeratios of their characteristic fragments. Ions of a molecule of interestare mass-to-charge selected by a mass selective ion optical device,along with other molecular ions of a closely similar mass-to-chargeratio. These ions are called the parent or precursor ions. These parentions are then fragmented using one or more processes, and the fragmentions are mass analysed - providing a so-called MS/MS mass spectrum.Molecules of different structure typically fragment to form differentfragment ions, and the parent molecules can be identified by studyingthe mass to charge ratios of those fragment ions. Where the fragmentmass spectra also contain interferences, or where a higher amount ofinformation than is present in MS/MS is required, further stages offragmentation may be used, producing MŜn mass spectra. Libraries ofprotein sequences have been developed and these are searched, usingalgorithms developed for the purpose, to match the fragment ion spectrato likely parent molecules.

This is a powerful and widely-used method in organic mass spectrometry.However it has certain disadvantages, relating to the requirement formore than one mass selective step. This requirement increases thecomplexity of the instrumentation required to perform the method, andincreases the time of analysis.

Besides using the technique of ion fragmentation to enable a parentmolecular ion to be identified, a high mass resolution mass spectrometermay be used to distinguish between molecular ions of closely similarmass to charge ratios. However, typically such high mass resolutionspectrometers are more costly and often very much slower (due to longermeasurement times) than their lower resolution counterparts.

If the fragment ion mass spectra are of high resolution and high massaccuracy, the match between the fragment ions and likely parentmolecules can be made with a higher degree of confidence. Consequentlyin order to identify large molecular ions most effectively, analystsoften use a combination of high resolution mass spectrometry andfragmentation methods. However combining the two methods results in aneven longer analysis time.

Methods such as those outlined above are routinely used for samplescontaining proteins. Typically the proteins are digested to producepeptides and these are ionised and introduced into the massspectrometer.

The target protein or mixture (for example a cell lysate) ispre-processed. Pre-processing can include filtering or cleaning. It isthen digested with a suitable cleavage reagent. The most frequently usedis the enzyme trypsin, but others, like Chymotrypsin, Cyanogen bromide,iodoso benzoate are also used. After digest and possibly cleaning themixture is fed to a mass spectrometer, usually following chromatographicseparation. Chromatographic separation usually limits the time availablefor the tandem mass spectrometry process. Chromatography times per peakrange from 30 seconds to less than 1 second with the trend being tofaster times.

Initially a full mass spectrum is taken, producing a so-called precursorion spectrum. Fragment ion spectra can be obtained for every ion speciesin the precursor ion spectrum (data-independent MS/MS). Alternatively, afrequently used approach is “data dependent” MS/MS. In this method, afull spectrum is acquired and afterwards the one or more most intensepeaks are selected, usually automatically, and subjected to MS/MSfragmentation, one by one. The precursor and fragment spectra arestored. Various enhancements to this include: temporary blacklisting ofprecursors to avoid re-measurement of intense ions; permanentblacklisting of precursors to avoid collection of MS/MS data of wellknown peptides or solvent components; whitelisting of masses of interestto allow fragmentation even when the most intense criteria are not met.However, there are two problems with this data-dependant MS/MS approach.Firstly, different runs of the same sample may produce very differentresults, because, for example, even small variations in peak heights inthe precursor ion spectra may result in different decisions beingautomatically made, leading to the selection of different precursor ionspecies for fragmentation. Secondly, in many cases there may notsufficient time to fragment all ions of interest within the time windowavailable due to the preceding chromatographic process.

The prior art data-dependant process in which two precursor ions areselected for MS/MS is shown as an example in the flow chart of FIG. 1.

After, or sometimes during measurement, the acquired data are evaluated.Many methods are known for this, such as (1) “de novo sequencing” inwhich the amino acid sequence is inferred directly from the spectra; (2)“sequence tagging” in which only part of the amino acid sequence isdirectly inferred from the spectra, and these small sections (“tags”)are used in a database search routine; (3) a direct database search isperformed just using the fragment ion spectra.

Database searching is performed to match fragments ions to their likelypeptide precursors. Automatic routines have been developed to performthe searches. The result is a list of likely precursors with a scoredenoting the confidence in the match. Optionally the database to besearched can be pre-selected by the user who can limit the search topeptide precursors known to be relevant, such as, for example, those foryeasts where the sample is known to have originated from a yeast.Optionally the computer search can also provide protein scorescalculated from the peptide scores to give an indication of the likelyproteins contained in the pre-digested sample. Typically the searchalgorithm returns a score-sorted list of the protein or peptidecandidates along with their scores. The interpretation is then typicallyleft to the user.

The standard approach is to submit a peak list of each of the MS/MSspectra together with the respective precursor mass (usually this is themass that triggered the MS/MS event in the data dependent setup) to a“search engine” for comparison with a database. Normally a check formore than one precursor in the mass selection window is not done. Manydatabases of proteins are publicly available. Some of them directlycontain proteins from previous analysis, others, such as SwissProt(http://expasy.org/sprot/), are computer translations of genomicsequences.

As the final goal of search engine use is to come up with one or moreproteins determined to be in the analyte mixture, the proteins in adatabase are “electronically digested” to peptides with propertiesmatching the cleavage reagent selected by the user. This “in silicodigestion” can happen on the fly or as an “indexing” step before theactual search is performed. All peptides matching the precursor masswithin a tolerance window defined by the user or inferred from the dataare considered “candidates”. Fragment ions from these candidates arethen predicted. Scores are associated with these candidates based on theMS/MS data, a higher score resulting when the MS/MS fragment ionspectrum contains the predicted fragments of the predicted candidates.

The prior art database search process is shown as an example in the flowchart of FIG. 2.

If deliberately or inadvertently more than one precursor ion species isselected at the same time for fragmentation, the fragment ion spectrumwill be more complex and the results from the database search enginewill be less accurate.

The prior art processes described in FIGS. 1 and 2 suffer from thedisadvantage that the time to obtain the score-sorted list of likelypeptides is slow, even where these data-dependant methods are used,because each precursor ion of interest alone must be selected andindividually fragmented, and the resultant ions mass analysedsequentially, before they can be processed using standard search enginetechniques. This is costly as instrument time is expensive, and it iswasteful as relatively large proportions of the sample (which may onlyexist in very small quantities) are consumed during the process.

One particular method of improving the throughput is described byMasselon and Smith in Analytical Chemistry, Volume 72, No. 8,pp1918-1924, 2000. In this method a form of multiplexing is performed.Fragment ions from more than one precursor are intentionally measured ina single mass spectrum taken with very high mass accuracy. The fragmention spectrum does then contain fragments from more than one precursorion species. This spectrum is sent to the database search engine asnormal, and the method relies on the high mass accuracy of the fragmentspectrum which enables most of the fragment ions to be attributed to aspecific parent polypeptide, though possibly not every fragment ionspecies can be assigned to a parent.

There are several disadvantages to the method of Masselon and Smith. Asnoted above, when fragment ion spectra from more than one precursor ionspecies are processed by the standard search engine methods, because thefragment ion spectra are more complex, the results from the databasesearch engine are less accurate, even though high mass accuracy has beenused. Furthermore, not only are the scores less accurate, a far greaternumber of false-positive identifications will result. Due to thecomplexity of the fragment ion spectra, the speed of the search engineis greatly reduced.

The present invention seeks to address these and other problems withprior art MS/MS data processing.

SUMMARY OF THE INVENTION

Against this background, and in accordance with a first aspect of thepresent invention, there is provided a method of identifying precursorion species from their fragments comprising:

-   -   (a) determining a quantity indicative of the mass of a plurality        of precursor ion species;    -   (b) fragmenting the ions of the plurality of precursor ion        species to form a plurality of fragment ions derived from the        plurality of precursor ions;    -   (c) mass analysing together/simultaneously, the fragment ions        derived from multiple precursor ion species;    -   (d) assigning one or more sample sets of multiple fragment ion        species to a particular one of the plurality of precursor ion        species, the or each sample set including fragment ion species        whose combined mass as determined in step (c)) corresponds with        that of the particular one of the precursor sample ion species        to which those fragment ion species are assigned;    -   (e) for one or more of the precursor ion species, forwarding        sample data identifying (i) the mass of the particular precursor        sample ion species identified in step (a), and (ii) the mass of        the multiple fragment ion species in the or each assigned sample        set for that particular precursor ion species, to a comparing        means, for comparison of quantities indicative of the mass of        the precursor and assigned fragment ion species with quantities        representative of the mass of ions in one or more reference sets        of reference fragment ion data and reference precursor ion data        respectively; and    -   (f) receiving, from the comparing means, information indicative        of the results of the said comparison, which has sought to        identify the precursor ion species to which the multiple        fragment sample ion species had been assigned.

Thus, multiplexing is again used as the method of improving throughput,and fragment ion data such as a fragment mass spectrum is obtained fromfragment ions derived from more than one precursor ion species. Both thefragment ion data and the precursor ion data are preferably obtainedwith high mass accuracy (eg <5 ppm, most preferable <2 ppm for thefragment and precursor sample ion data, with a resolving power of100,000 at FWHM). However, instead of utilising the database searchengine on this resultant fragment ion data, it is instead furtherprocessed. In this additional processing step, the fragment ion data issearched for multiple fragments whose combined mass matches that ofprecursor ion masses found in the precursor ion data, within certainaccuracy limits. The accuracy limits may for example be determined fromthe mass accuracy of the fragment and parent ion data. Having matched aset or sets of multiple fragment ions to precursor ion species, thefragment ion data are broken down into portions, one portion for eachprecursor ion species, and containing only the set(s) of fragment ionspecies assigned to that particular precursor ion species. This processeffectively allows the reconstruction, in preferred embodiments, ofsimplified fragment sample ion spectra from precursor ion species. Onefragment ion spectrum may for example be produced for each precursor ionspecies, as though MS/MS spectra had been obtained for each precursorion species one at a time. This process deconvolutes the multiplexingprocess, yet retains all the speed advantage gained by the multiplexingprocess. The resultant sample sets of fragment ion data (for example,deconvoluted fragment ion spectra) are then preferably sent one by oneto the database search engine, which performs the standard databasesearch on each, giving, in preferred embodiments, a score-sorted list oflikely candidates for each deconvoluted fragment ion spectrum.

The method of the present invention thereby greatly improves theaccuracy of the results from the database search engine. It also greatlyimproves the speed of search.

By the term “analysing together/simultaneously”, it is meant that themethod involves searching through the fragment ions from more than oneprecursor sample ion (whether those fragment ions are created at thesame time, by simultaneous fragmentation of multiple precursors, or byaccumulation together of fragments from sequential fragmentation of oneor more precursors) at the same or substantially the same time. Moreparticularly it is not intended to imply that the actualdetection/identification of the fragment ions takes place as a singleevent. Whilst in the case of certain types of mass spectrometry such asFT-ICR or Orbitrap™ MS, the fragment ions are detected together, inothers, such as TOF-MS, the ions are ejected sequentially to a detectorinstead. Nonetheless the analysis itself (prior to detection) is carriedout on fragment ions from more than one precursor, in tandem to permitthe above mentioned multiplexing.

Moreover, it will be understood that, although some preferredembodiments will determine the mass (or even the mass to charge ratiom/z) of the precursor and/or fragment ions, this is not essential to thesuccessful operation of the invention. Many different physicalparameters such as (but not limited to) time of flight, frequency,voltage, magnetic field deflection etc. might be measured (dependent forexample on the chosen method of ion detection), each of which is relatedto or allows derivation of the ion mass or m/z. However it is notnecessary that the mass itself is calculated in each case; it may becomputationally more efficient not to convert measured parameters in anon-mass space into mass. Furthermore the quantity stored in thecomparing database may itself not be held as a mass but instead adifferent quantity related to mass. Thus the term “a quantity indicativeof mass” is to be interpreted broadly to encompass mass and otherquantities.

Preferably, the method comprises assigning one or more pairs of fragmentions to a particular precursor ion species. This may be on the basis ofa combined mass of the two fragment ion species corresponding with themass of that assigned precursor ion species, by having a total mass thatadds up to the mass of the precursor on species, or otherwisecorresponds by having a predetermined offset mass from that precursor(eg as a result of neutral loss of water molecules duringfragmentation). The pairs of assigned fragmentations may be so-called‘golden pairs’ of ions as identified via different fragmentationtechniques.

The method of the present invention also reveals ion species in thefragment sample ion data that cannot be assigned to precursor ionspecies in the precursor ion spectrum. These fragment ion species may ormay not then be sent to the database search engine. If sent, they may besent alone, and will not then contribute to the complexity of the otherdeconvoluted fragment sample ion data in the sample sets, as they wouldin prior art methods.

The invention may also be used to gain the speed and accuracy advantageswith MS/MS/MS techniques, or MSn. Since the prior art multiplexingarrangement of Masselson and Smith in fact requires high mass accuracyfor both the precursor and fragment ions, the method of embodiments ofthe present invention has no additional time penalty relative to thatart (in terms of data collection), whilst it does in contrast providesignificantly more accuracy. Of course, relative to previous methodsthat did not attempt to multiplex precursors, embodiments of the presentinvention provide for significant time savings.

Further features and advantages of the present invention will beapparent from the appended claims and the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow chart of a prior art data dependent process forselecting two precursor ion species for MS/MS analysis;

FIG. 2 shows a flow chart of a prior art database search procedure;

FIG. 3 shows, in block diagram form, an overview of one exemplary massspectrometer suitable for implementing the method of embodiments of thepresent invention;

FIG. 4 shows, functionally and schematically, a second exemplary massspectrometer suitable for implementing the methods of embodiments of thepresent invention;

FIG. 5 shows, functionally and schematically, another exemplary massspectrometer suitable for implementing the method of embodiments of thepresent invention;

FIG. 6 shows still a further exemplary mass spectrometer, in functionaland schematic form, likewise suitable for implementing the method ofembodiments of the present invention

FIG. 7 shows a part of a mass spectrum for the molecule C22H42N9O6 andits fragments;

FIG. 8 shows a plot of a figure of merit for accuracy of identificationof precursor ion species from experimental fragment data, as a functionof the number of multiplexed sample fragment data sets, when employing aprior art method and a method in accordance with the present inventionrespectively;

FIG. 9 shows a distribution of experimentally determined figures ofmerit for accuracy of identification of 1000 MS/MS spectra, whenobtained individually and when obtained by multiplexing groups of 4fragment sample ion data sets together in accordance with an embodimentof the present invention; and

FIG. 10 shows a flow diagram of a procedure that employs the method ofembodiments of the present invention, for multiple stages offragmentation (MSn).

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Preferred embodiments of the present invention provide for a method ofidentifying precursor ion species from their fragments. Whilst themanner in which the fragment ions are produced is not in itself critical(and indeed, optionally, different fragmentation techniques and energiesmight be employed on the same precursor ions to obtain differentfragment ion species), nonetheless one suitable technique for thefragmentation of precursor sample ions and the collection of massspectrometric data from such a process will first be described so as topermit of a better understanding of the invention. It is nevertheless tobe stressed that the following description of a preferred embodiment ofan arrangement for fragmenting precursor ions represents merely one ofmany different ways of so doing, and moreover the manner in which theions are detected is likewise capable of implementation in a variety ofdifferent ways.

Referring first to FIG. 3, a mass spectrometer is shown. The massspectrometer 10 comprises an ion source 20 for generating ions to bemass analysed. The ion source 20 shown in FIG. 3 may be a pulsed lasersource (preferably a matrix-assisted laser desorption ionization (MALDI)source in which ions are generated through irradiation from a pulsedlaser source 22), a continuous ion source, such as an atmosphericpressure electrospray source, or otherwise.

The ions from the ion source 20 are admitted into an ion trap 30 whichmay, for example, be a gas-filled RF multipole or a curved quadrupole asis described, for example, in WO-A-2005/124821 and more recently inWO-A-2008/081334 whose contents are incorporated by reference.

The ions are stored in the ion trap 30, and collisional cooling of theions may take place as is described for example in our co-pendingapplication number WO-A-2006/103445, the contents of which are alsoincorporated herein by reference. Storage takes place in the ion trap 30until the RF is switched off and a DC voltage is applied across therods. This technique is set out in detail in our co-pendingapplications, published as GB-A-2,415,541 and WO-A-2005/124821, thedetails of which are incorporated herein in their entirety.

In a first cycle, a range of precursor ions (either contiguous across arange of m/z or a series of non-adjacent masses) is ejected from the iontrap 30 to a mass analyser 70 such as an Orbitrap, FT-ICR or other highmass accuracy analyser. This produces a high mass accuracy precursorsample ion mass spectrum for the precursor ions ejected from the iontrap 30 in the first cycle. The precursor sample ion mass spectrumserves several purposes. Firstly it may be utilized to identify a subsetof precursor ions to be analysed (since not all precursor ions arelikely to be of analytical interest). Secondly by obtaining theprecursor spectrum at high accuracy, a measured precursor mass peak maybe sent for analysis along with the fragment ion data, as explainedfurther below.

In a second cycle, the ion trap 30 is refilled from the ion source 20.Again the ions are cooled. This time, however, rather than massanalysing all of the precursor ions together, individual precursor ionsare identified for further analysis, from the previously obtainedprecursor sample ion mass spectrum. To isolate such identified precursorions, the contents of the ion trap 30 are pulse-ejected towards an ionselection device which is preferably an electrostatic trap 40. Pulsedejection produces narrow ion packets. These are captured in theelectrostatic trap 40 and experience multiple reflections therein as isdescribed in our copending application GB0725066.5 and WO-A-2007/122378.

Ejection from the ion trap 30 to the electrostatic trap 40 occurs viaion optics (not shown in FIG. 3), with optional control of the number ofions to avoid subsequent problems with space charge.

After acceleration through the ion optics the ions are focused intoshort packets between 10 and 100 ns long for each m/z and enter theelectrostatic trap 40. On each reflection in the electrostatic trap 40,or after a certain number of reflections, unwanted ions arepulse-deflected out of the electrostatic trap 40, for example to adetector 75 or to a fragmentation cell 50. Preferably, the ion detector75 is located close to the plane of time-of-flight focus of the ionmirrors, where the duration of the ion packets is at a minimum. Thus,only ions of analytical interest are left in the electrostatic trap 40.Further reflections will continue to increase the separation betweenadjacent masses, so that further narrowing of the selection window maybe achieved. Ultimately, all ions having a mass-to-charge ratio adjacentto the mass-to-charge ratio m/z of interest are eliminated, leaving thesingle precursor sample ion species in the trap, which was identifiedfrom the precursor mass spectrum obtained in the first cycle ofanalysis.

That single precursor sample ion species in the electrostatic trap isthen ejected to a fragmentation cell 50. Preferably, the fragmentationcell 50 is a segmented RF-only multipole with axial DC field createdalong its segments. The selected precursor sample ions are ejected fromthe electrostatic trap 40 to the fragmentation cell 50 with sufficientenergy to allow them to fragment within the fragmentation cell 50.

Following fragmentation in the fragmentation cell 50, ion fragments fromthe first precursor ion species are transferred to an auxiliary ionstorage device 60. Here they are stored whilst subsequent cycles takeplace, as described below.

Once the fragment ions from the first precursor sample ion species havebeen stored in the auxiliary ion storage device 60, the steps arerepeated for a second precursor sample ion species. Specifically asecond precursor sample ion species (again preferably selected basedupon the precursor sample ion mass spectrum previously obtained) isisolated in the electrostatic trap 40 and then sent to the fragmentationcell 50, and fragmented, with the fragments being passed as in theprevious cycle then to the auxiliary ion storage device 60 where thefragments from the second precursor sample ion being stored along withthe fragments from the first precursor sample ion there.

Further cycles as above may be carried out subject to the limits of dataprocessing (for a discussion of which, see below), subject to spacecharge limitations, and subject to a total ion storage time for themultiple fragment ions in the auxiliary ion storage device 60.

Once the multiple fragment ions have been accumulated in the auxiliaryion storage device 60, they are ejected back to the ion trap 30 wherethey enter it via a different orifice to the one from which theirprecursors were originally ejected as is described in detail in theaforementioned WO-A-2007/122378. From here they are ejected to the highmass accuracy mass analyser (eg Orbitrap) 70 for mass analysis. Once themass analysis is complete, the data obtained from mass analysis of theprecursor ions is processed along with the data obtained from a massanalysis of all of the fragment ions together, in a manner to bedescribed below. The processing may take place either locally, forexample in the processor of a local computer that controls or is linkedto the mass spectrometer 10 (not shown), may be stored locally forsubsequent analysis, and/or may be sent as one or more data files to aremote location for subsequent processing there, with the results ofthat processing being returned to the user of the mass spectrometer 10subsequently.

The foregoing describes the capture of mass spectrometric data from aplurality of precursor sample ions in a first cycle, and then, byisolating each precursor ion species (identified from that precursorsample ion mass spectrum as being of interest) in successive cycles,accumulating the totality of the fragment ions from each precursor ionspecies by storing them together for simultaneous/parallel analysis oftheir fragment mass to charge ratios. However it will be understood, ofcourse, that this is merely one way in which multiple precursor andfragment ions can be analysed at once using the techniques to bedescribed below. For example, rather than isolating individual precursorspecies and then accumulating these together for fragmentation, allprecursor ions may be isolated together in one step, for example usingthe procedure described in WO-A-2008/059246 the contents of which areincorporated by reference in their entirety.

The selection of precursors can be achieved in many different ways,which can be classified as data dependent or data independent. Forexample, in a data independent mode, a contiguous mass range may beselected (which may or may not include a plurality of ion species).Alternatively, a non-contiguous mass range may be selected, that is,precursors from a plurality of non-adjacent mass windows may beselected. In a data dependent mode, a predetermined number of precursorion species may be selected (eg 4), and these may be sorted by intensityfor example. “Inclusion” and “Exclusion” lists may be employed forprecursor picking (which lists will be familiar to those skilled in theart), and these may optionally be dynamic lists. Other precursoridentification criteria may be employed, eg Kendrick mass offset (“massdefect”), neutral loss for MS3, and so forth. Finally it may be possibleto select precursors initially on the basis of certain criteria and thento carry out an additional “safety” MSn scan of the precursor ions thatremain.

In terms of how the multiplexing is achieved, it will likewise beunderstood that the method is equally applicable both to serial analysisand fragmentation of single precursor ions (with all of the fragmentsbeing collected together in the auxiliary ion storage device 60 asdescribed above), and to parallel analysis of multiple precursor ions(whether selected in a single cycle, or by accumulation in for examplethe auxiliary ion storage device 60 in multiple successive cycles), byfragmentation of the multiple precursor sample ion species together andparallel analysis of the multiple fragment sample ion species therebyproduced.

Likewise, although it is desirable that the mass analysis of bothprecursor and fragment ions be carried out to a high mass accuracy, thiscan nevertheless be achieved at various locations and in various wayswithin the exemplary arrangement of FIG. 3. For example, precursor ionsstored in the ion trap 30 may be mass-analysed in the electrostatic trap40, by ejecting the mass from the ion trap 30 to the electrostatic trap40, isolating the precursor ions there and ejecting them to the detector75, rather than passing them from the ion trap 30 to the Orbitrap orother mass analyser 70. By way of example only, the detector 75 may bean electron multiplier or a microchannel/microsphere plate which hassingle ion sensitivity and can be used for detection of weak signals.Alternatively, the detector may be a collector and can thus measure verystrong signals (potentially more than 104 ions in a peak). More than onedetector could be employed, with modulators directing ion packetstowards one or another according to spectral information obtained, forexample, from the previous acquisition cycle. In this way, high massaccuracy data from the precursor sample ion species may be obtained viathe electrostatic trap 40. Moreover, it will be understood that themanner of detection is also dependent upon the nature of the massanalysis technique being employed. For example, if time of flight massanalysis is being carried out, then ions of increasing m/z are typicallydetected sequentially in time via a microchannel plate for example. Ifon the other hand an Orbitrap or FT-MS analysis is being carried out,simultaneous detection of substantially all ions (via a time domaintransient), followed by a subsequent Fourier transform into thefrequency domain, may be carried out instead. From this, in turn, ionmasses may be determined. It will thus be understood that mass itselfneed not be determined from the ion detection; time (of flight),frequency, voltage, magnetic field and other physical parameters may bethe primary measured quantity and it is not necessarily essential thatthose primary measurement parameters are converted into ion mass. It mayinstead be computationally effective to bypass calculation of ion massesand do some of the subsequent analysis (to be described further below)directly in the space of the originally detected quantity. Thus, in thefollowing, although the term “mass” (or mass to charge ratio) isemployed, it is to be understood that in fact the calculations might becarried out on quantities merely related to, and not directlyrepresentative of, ion mass. Also many mass spectrometers anyway detectmass to charge ratios of ions. Various known methods exist fordetermination of a molecular mass from this measured m/z (see eg U.S.Pat. No. 5,072,115 and Hort et al in J Am Soc Mass Spectrometry, 2000,11, 320-332). Most of the calculations described below are mostconveniently carried out in the mass space, where possibly the chargecarrying adducts are already corrected for. The necessarytransformations are anyway well known in the art and/or may be readilyascertained.

Having described one exemplary way of obtaining the mass spectrometricdata from a plurality of precursor sample ions and their fragments, amethod embodying the present invention, which involves the processing ofthat data in parallel (multiplexing) so as to permit of identificationof multiple precursor sample ions (or derivatives/parents thereof)substantially simultaneously, will now be described.

The composite high mass accuracy fragment sample ion mass spectrum thathas been obtained, and the precursor mass spectrum, are both firstlyde-charged and de-isotoped to produce simplified spectra. The fragmentmass spectrum is then scanned to identify pairs of fragments whosecombined mass matches the mass of a one of the precursor ion species.Complementary pairs of fragment ions have been found to possess uniquespecificity among all types of fragments generated through CollisionallyActivated Dissociation (CAD), although other forms of fragmentation canequally be employed.

Although both the precursor and the fragment ion masses are measured tohigh mass accuracy, nonetheless they will still be subject to a degreeof error as a consequence of the finite accuracy of the massmeasurement. This measurement error may be used to inform the processingof the fragment mass information: a match may only be identified whenthe combined mass of the two fragment ions is the same as that of a oneof the precursors to within a predetermined margin of error (or is thesame as a fixed, predetermined offset from a precursor, as a result of aneutral loss of H₂O etc).

Having matched pairs of fragment ions to precursor ion species, the(composite) fragment ion spectrum is broken down into portions, oneportion for each fragment pair, and containing only each fragment pair.The analysis of the composite fragment mass spectrum is continued untilno further pairs are identified to within the stipulated accuracylimits. Any single fragment ions left unassigned to a precursor ionspecies can be discarded or, included but ignored in subsequentidentification analysis (described below).

Once the composite fragment spectrum analysis has been completed, one(simplified) fragment ion spectrum is reconstructed for each precursorsample ion, respectively, by stitching together or otherwiseconcatenating each single broken down portion of the composite fragmentspectrum, for all pairs of fragment ions that have been linked to aparticular precursor sample ion species. In other words, for ‘n’precursor sample ions being analysed together, with a composite fragmentsample ion mass spectrum obtained by fragmenting those ‘n’ precursor ionspecies (either concurrently or sequentially but with all fragmentsanalysed together), ‘n’ separate simplified fragment mass spectra(containing only data from pairs of fragments having the same combinedmass as a particular one of the ‘n’ precursors) will result from theabove analysis.

The resultant deconvoluted fragment ion spectra are then sent one by oneto a search engine such as Mascott™ or Sequest™ along with the measuredmass to charge ratio of the associated precursor sample ion species. Thesearch engine carries out a standard database search on each syntheticfragment spectrum, and returns a scored (and optionally score-sorted)list of likely candidates for each such deconvoluted (synthetic)fragment ion mass spectrum. Although identification of precursor ionsbased upon the mass of the synthetic fragment mass spectra alone iscurrently preferred, nonetheless the (relative) abundance of each mayalso be employed, optionally, to assist further with identification.

This technique deconvolutes the multiplexing process yet retains all ofthe speed advantage that is gained by this multiplexing process: in theprior art technique of Masselson and Smith, in fact the most accurateresults are obtained when the precursor ions are accurately identifiedin the precursor mass spectrum and to do this it is desirable that theprecursor ions are analysed at high mass resolution (and for the priorart method to work at all, it is as already discussed necessary formaximum mass accuracy of the fragment ions). In other words, embodimentsof the present invention do not introduce any additional time penaltyrelative to the Masselson and Smith method, and provide significant timeadvantages over non-multiplexed MS/MS techniques. Furthermore,embodiments of the present invention do not result in a significantreduction in the accuracy of the results relative to non multiplexingtechniques. In contrast to the multiplexing technique of Masselson andSmith, however, in the method now described there is no fatal drop offin the accuracy of identification of the fragment spectra as the numberof multiplexed precursors increases above 2.

To illustrate further the principles described above, and in particularthe method of matching fragment ion masses to a precursor ion mass, FIG.7 shows a part of a mass spectrum for the molecule C₂₂H₄₂N₉O₆ and itsfragments. In accordance with the techniques described above, firstlythe precursor ion species (C₂₂H₄₂N₉O₆) is adduct corrected (the adductis H+ with a (known) mass of 1.007825 amu). The adduct corrected mass pof the precursor is hence 528.32526−1.007825=527.317984 amu.

Next, a first fragment peak (identified in Figure as B₁[R]) is selectedand its measured mass is again adduct corrected. The (corrected) mass M₁is stored (in FIG. 7, it is noted as 157.10839−1.007825=156.101114).Next all other peaks are searched for a mass M₂ which, with adductcorrection, has a mass M2 (=M₂′−1.007825) such that M₁+M₂=p. Once M₂ isidentified, M₁ is placed in a list of verified fragment masses. Theprocess is repeated for other fragment ions. Tables 1 and 2 show theuncorrected and adduct corrected results for the fragment ions [P] [R],[RK], [QP], [RKQ] amd [KQP] respectively (the molecular structure isshown in FIG. 7 for each fragment ion but is omitted here for brevity).It will be seen that, in each case, the pairs of adduct correctedfragment ion masses add up to the precursor ion mass when adductcorrected.

Once the list of verified fragment masses has been compiled, it may besubmitted (along with details of the precursor ion mass) for furtheranalysis, for example by a search engine as previously described.

TABLE 1 uncorrected parent and fragment ion masses Fragment Mass ofFragment Mass of Parent Mass of Name 1 Fragment 1 Name 2 Fragment 2 Nameparent [R] 157.10839 [KQP] 372.22415 [RKQP] 528.32526 [RK] 285.20335[QP] 244.12918 [RKQP] 528.32526 [RKQ] 413.26193 [P] 116.07061 [RKQP]528.32526

TABLE 2 adduct corrected parent and fragment ion masses Fragment Mass ofFragment Mass of Parent Mass of Name 1 Fragment 1 Name 2 Fragment 2 Nameparent [R] 156.101114 [KQP] 371.216874 [RKQP] 527.31799 [RK] 284.196074[QP] 243.121904 [RKQP] 527.31799 [RKQ] 412.254654 [P] 115.063334 [RKQP]527.31799

FIG. 4 shows a functionally schematic diagram of a preferred massspectrometer arrangement for implementation of preferred embodiments ofthe present invention. In FIG. 4, optional sample preparation firsttakes place at sample preparer 5. Chromatography (and, in particular,liquid chromatography LC) then takes place at stage 15 and the resultantmolecules are ionised in an ion source 20. A first set of ions is thenselected from these, in an ion selector 25. Following selection, ionsare fragmented in a collision cell 50 and then collected downstream ofthere, in an ion collector 35.

The process of selection in the ion select 25, fragmentation in thecollision cell 50, and collection in the ion collector 35 is repeateduntil the desired combination of ions is present in the ion collector35. After that, ions are ejected to a mass analyser 45 (which may, forexample, be an Orbitrap™ mass analyser) and the output of the massanalyser 45 is processed in a data processing system 55. The stepscarried out in the data processing system 55 are as outlined above andinclude the steps of deconvolution of the fragment ions to separate datasets for the respective parent ions, as well as optional database searchor sequencing.

Optional feedback from the data processing system can be used further tocontrol the ion selection and fragmentation processes.

It is to be understood that the arrangement of FIG. 4 is intended to bea functional representation of the preferred components of a massspectrometer system for implementing embodiments of the presentinvention. Different operational stages could be executed in a singlehardware element, so, for example, the steps of selection, fragmentationand collection could all be executed in a single ion trap such as thelinear trap quadrupole (“LTQ”) linear ion trap of an LTQ fouriertransform ion cyclotron resinence (LTQ FT ICR) mass spectrometer, withonly the accurate mass analysis being done in a separate mass analysingapparatus. Theoretically, even the mass analysis could be done in thesame ion trap—see, for example, U.S. Pat. No. 4,755,670. Also it is tobe understood that the selection of multiple ions need not besequential. A suitable wave form, such as is described for example inU.S. Pat. No. 4,761,545, can be used to select all desired ionssimultaneously in an ion trap. Similar concepts exist for mass filters.

Turning now to FIG. 5, a functionally schematic representation of analternative mass spectrometer arrangement is shown, and which issuitable for implementation of preferred embodiments of the inventionagain. As with the arrangement of FIG. 4, sample molecules may beprovided by optional sample preparation apparatus 5, coupled to a liquidchromatography arrangement 15 which supplies sample molecules to an ionsource 20.

As seen in FIG. 5, the ion source 20 provides ions to a mass selectionquadrupole Q 27: from there, selected ions pass to a collision cell q50, and from there to a quadrupolar ion collector Q 29. Downstream ofthe quadrupolar ion collector 29 is an optional time of flight massseparator 47 connected to data processing system 55.

In typical operation of the arrangement of FIG. 5, a “normal” massspectrum is acquired, either by scanning of the mass selectionquadrupole 27, or by collection of ions in the quadrupolar ion collector29, followed by a mass selective scan onto a detector, or by collectionof ions in the quadrupolar ion collector 29 followed by mass analysis inthe time of flight analyser 47.

Optionally, a decision about the following mass steps for analysis isbased upon the previously acquired spectrum, although this procedure isof course not necessary when the final goal is to have all ionsfragmented.

Next, the mass selection quadrupole Q 27 is operated to select thedesired precursor masses or mass ranges one after another. The ionswhich pass through the mass selection quadrupole Q 27 are thenfragmented in the collision cell q 50 and the resultant fragments arecollected either directly in that collision cell q 50, thus obviatingthe need for the subsequent quadrupolar ion collector 29, or in thatquadrupolar ion collector 29.

The resultant fragments are mass analysed in the time of flight analyser47. Data processing, as described previously and in accordance withembodiments of the present invention, is then applied to the acquiredmass information.

FIG. 6 shows, again functionally schematically, still anotherarrangement of a mass spectrometer suitable for implementing embodimentsof the present invention. In FIG. 6, once again optional samplepreparation and liquid chromatography steps may be carried out so as toprovide sample molecules to an ion source 20. Ions from the ion source20 are then directed towards a linear ion trap 26. Downstream of thelinear ion trap is an ion collector 31, communicable with an ionfragmentation means 50′ (which may be a collision cell) and also,separately, with an Orbitrap™ trapping mass analyser 70. The Orbitrap™mass analyser 70 is connected to data processing system 55.

The arrangement of FIG. 6 offers multiple modes of operation. In a firstmode, following a normal mass scan, a precursor ion is selected andfragmented in the linear ion trap 26. The resultant fragments are thensent to the intermediate ion store 31. The next precursor ions aretreated the same way and injected into the intermediate ion store 31 tobe stored alongside the previously stored fragment ions. Once alldesired fragments from the different precursor ions have been collectedin the intermediate ion trap 31, they are sent together to the Orbitrap™orbital trapping mass analyser 70, for mass analysis and detection.Processing takes place at the data processing system 55 in accordancewith previously described principles.

In an alternative mode of operation of the arrangement of FIG. 6,multiple precursor ions are selected at the same time, for example, witha stored waveform inverse fourier transform (SWIFT) excitation of someother form of “notched” waveform in the linear ion trap 26. The ions arethen fragmented together, either in the linear ion trap 26, for exampleby collision induced dissociation (CID) or electron transferdissociation (ETD), or in a separate fragmentation means 50′ where highenergy collision activated dissociation (HCD) may occur, thefragmentation means 50′ being accessed via the intermediate ion trap 31.Fragments are then sent back out of the ion fragmentation means 50′ andcollected in the intermediate ion store 31. After that they may beinjected into the Orbitrap™ 70 for analysis.

In still a further mode of operation of the arrangement of FIG. 6, thefirst and second modes described above can be combined. For example, amass range may be isolated or different mass ranges may be isolated andadded.

In yet a further mode of operation of the arrangement of FIG. 6,sequential precursor ion selection in the linear ion trap 26 may takeplace, with transfer to the intermediate ion store 31, followed byfragmentation of all ions together in the ion fragmentation means 50′.The resulting fragment ions are then collected in the intermediate ionstore 31 again and mass analysed in the Orbitrap™ 70.

In each case, of course, once the mass data has been obtained, it can beprocessed using the data processing system 55 in accordance withpreviously described principles embodying the present invention.

Actual data from an MS/MS experiment were obtained using the methodembodying the present invention. The precursor and fragment spectra wereboth obtained with high mass accuracy (2 ppm mass accuracy at 1 Sigma),using an FT-ICR mass analyser and with a mass resolving power of 100,000full width at half maximum (FWHM). The database search forming thelatter stages of the method was carried out with thresholds of 5 ppm (3Sigma) and 10 ppm (6 Sigma) using the Mascot search system.

FIG. 8 shows a plot of the average Mascot score as a function of thenumber of precursor ions and their fragment spectra that have beenmultiplexed together, using the data thus obtained and applying themethods of the present invention. For comparison, the average Mascotscore obtained using the prior art Masselson and Smith technique isshown (they in fact employed the Sequest system which is similar to theMascot system for database searching purposes).

Both methods yield reducing scores with increasing number of multiplexedpeaks, but for the prior art method, the predicted score (using Mascot)falls dramatically even with only two multiplexed peaks. A score ofaround 30 is usually considered acceptable. It can be seen from FIG. 8that the method of embodiments of the present invention enables manymore precursor ion species to be multiplexed than the prior art methodsfor the same acceptable score. This results in a far greater improvementin throughput. It may also mean that far more useful information can beobtained about a sample in the time window available from thechromatographic separation which often precedes the mass spectrometry.

To obtain MS/MS data from 4 precursor ion species using a high massaccuracy spectroscopic technique, such as FT-ICR-MS or Orbitrap MS,which require ˜0.5 seconds per spectral acquisition, the time taken toindividually select, fragment and mass analyse is 2.5 seconds, beingmade up of one precursor ion spectrum and 4 fragment spectra. The timeusing the present invention is only one second, being made up of oneprecursor ion spectrum and one fragment ion spectrum. A factor 2.5time-reduction is achieved with Mascot predicted scores staying wellabove the acceptable level of 30.

As noted above in relation to FIG. 8, the method of the presentinvention does suffer from a reducing Mascot score with increasingnumber of precursor ion species multiplexed, but this reduction ismodest. To further illustrate this, direct comparison withnon-multiplexed results has been made. One thousand mass spectra wereselected from a database of CAD MS/MS spectra. Groups of four spectrawere summed together to simulate the multiplexed fragment ion spectrumthat would have been obtained in each case had the four precursor ionspecies been fragmented and the fragments combined and mass analysedtogether. This resulted in 250 simulated multiplexed MS/MS spectra. Theprocessing method of the present invention was then followed, and theresults compared to those obtained without multiplexing.

Following the method embodying the present invention as described above,each simulated multiplexed fragment mass spectrum was de-isotoped,de-charged, and converted to a list of neutral fragment masses. For eachof the four precursor masses Mm, pairs mi and mj of fragment masses wereselected, so that mi+mj=Mm within a mass uncertainty of ±15 mDa, whichis related to the mass accuracy of the original spectra. Thus eachsimulated multiplexed mass spectrum was separated into four deconvolutedMS/MS spectra.

The original 1000 MS/MS spectra were submitted to Mascot, which resultedin 980 above-threshold peptide identifications. The remaining 20 massspectra (0.2%) were not identified mainly because the protein databasehas changed after the identified spectra were put in the MS/MS database.The distribution of Mascot scores is shown in FIG. 9 by black columns.The 1000 deconvoluted mass spectra were also submitted to Mascot. Intotal, 899 peptides were identified (91%). The resultant distribution ofMascot scores is shown in FIG. 9 by grey columns. Sequences of only 2deconvoluted peptides out of 899 did not coincide with the normallyidentified sequences, corresponding to 0.22% false positive rate.

Based upon the use of CAD to fragment the precursor ions and detectionof ions in an Orbitrap mass analyser, it is possible to estimate athroughput improvement relative to a non-multiplexed MS/MS experiment.If carrying out CAD without fragment detection takes 1 time unit andOrbitrap detection takes 4 time units, then the overall time taken for a1 in 8 cycle in “normal” (non-multiplexed) mode is 1×4 (MS) plus 8×4(MS/MS)=36 time units. In multiplexing mode, the time taken is 1×4(MS)plus 2×4 (MS/MS)=12 time units. Thus, there is approximately a threetimes throughput increase relative to a non multiplexed technique, withminimal reduction in accuracy of identification of sample ion species.

Various modifications, alternatives and additions to the techniquesdescribed above are envisaged. For example, to assist further with theprocess of matching pairs of fragment ion species to their precursor,the following methods may also be used.

(1) Before de-isotoping the precursor and fragment ion spectra, the finestructure of isotopic peaks may be noted, for example the presence of 13C or 32 S. Such isotopes in the precursor species will also be observedin their corresponding fragments. This can be used to confirm ordisprove assignments or help identify precursor-fragment relations thatcannot be identified by addition of fragment pairs alone.

(2) Directly assign fragments to certain precursors when their mass fitsonly one of the precursors, for example when the fragment is too massiveto have come from a lower mass precursor.

(3) Use accurate mass information, optionally together with informationon substance class (e.g. knowledge that the sample is a peptide) toexclude/include certain fragments. Some fragments can only be from acertain precursor just by their accurate mass, the precursor accuratemass and the possible choice of neutral losses.

(4) Perform the deconvolution whilst sample analysis proceeds. Possibleinterferences may be identified and resolved in the next cycle ofanalysis of the same sample, by including the unresolved precursor ionspecies a second time. This subsequent cycle will produce a differentmultiplexed fragment spectrum as all but one precursor ion species willbe different. Identification of the fragments of the previouslyunresolved precursor ion species can be attempted on this data set or ona combination of this and the previous set.

The foregoing describes a technique for multiplexed analysis ofprecursor and fragment ions in MS/MS experiments. However, it is to beunderstood that the invention is not limited to single stagefragmentation. In particular the methods described above are equallyapplicable to MS³ or even MS^(n) experiments.

FIG. 10 shows a flow chart illustrating how MS3 may be carried out and,in particular, how the method of embodiments of the present inventionmay be applied both to first and second generation (“grandchildfragments”) fragment ions. At step 100, as previously described inconnection FIG. 3, a precursor mass spectrum is obtained to highaccuracy, as is MS/MS. At step 200, the precursor ion species ofinterest are accumulated together either by multiple cycles whichisolate different precursor ion species in the electrostatic trap 40, oralternatively by selecting narrower “windows” of multiple precursor ionspecies in the electrostatic trap 40. The accumulated precursor ionspecies are then fragmented together (step 300) in the fragmentationdevice 50 and the multiple fragment ion species from the multiple ioncursor ion species are accumulated together in the auxiliary ion storagedevice 60, step 400. As an alternative to the accumulation of all of theprecursor ion species of interest together before fragmentationaltogether, instead the precursor ion species can be isolated one at atime, fragmented individually, but still with the fragment ions fromeach precursor ion being accumulated together, again as previouslydescribed.

Next, at step 500, a mass spectrum of the fragments is obtained via themass analyser 70, to high mass accuracy. The obtained mass spectrum ofthe fragments is sent for processing at steps 600 and 700, to bedescribed below.

Next, in a first loop, a further set of precursor ions is accumulated(step 200 again). These are fragmented together (step 300) to form anaccumulated set of first generation fragment ions which are stored inthe auxiliary ion storage device 60 (step 400 again). This time,however, instead of obtaining a mass spectrum of these fragments, theyare instead returned from the auxiliary ion storage device 60 back tothe fragmentation device 50 where they are fragmented once more. This isshown as step 800 in FIG. 10. The resultant second generation fragmentions (grandchild fragments) are, at step 900, then mass analysed bysending them to the mass analyser 70 via the ion trap 30.

The method described above which embodies the present invention isapplied to the first generation mass spectrum obtained at step 500, soas to assign fragment ions to precursor ion species (step 600). Theresults of that assignment are stored at step 700. Likewise, the massspectrum of the second generation (grandchild) fragment ions is analysedusing the technique of embodiments of the present invention so as toassign second generation fragment ions to first generation fragment ionspecies. This is shown at step 1000. Again the results of that analysisare stored at step 700.

Applying techniques of the present invention to multiple stages of massspectrometry (MSn) provides for a potentially very significant timesaving relative to the prior art. The step 100 of obtaining a spectrumof the precursor ions takes approximately 0.5 seconds. Obtaining themass spectrum of the first generation fragment ions (step 500) likewisetakes 0.5 seconds, and it may even be possible to dispense with thisstep entirely when MS3 is being employed. Finally, the mass spectrum ofsecond generation fragment ions at step 900 takes about 0.5 seconds.

Thus, at worst, the total data collection time is 1.5 seconds. The priorart techniques take at least 10.5 seconds because the four separatefragment ion spectra take approximately 2 seconds to obtain and thesixteen consequential second generation fragment spectra take 8 secondsin total.

Clearly, the technique becomes more complicated as further generationsof fragments might be obtained but, equally, the time saving becomeshigher. One of the purposes of MS3 experiments is to disambiguateneutral fragments such as water, ammonia, phosphorylation or other sidechain losses, and loss of sugars from glycopeptides.

Furthermore, although the foregoing describes the analysis of fragmentions generated through CAD, it is to be understood that the techniquesare equally applicable to many other forms of ion fragmentation such as(but not limited to) ECD, ETD, metastable ion bombardment, CID (bothtrap CID and HCD), and IRMPD, for example. Indeed, as yet anothervariation to the methods described above, and in order to yield furtherinformation, the isolation of precursor ion species and the subsequentfragmentation may be repeated but with the fragmentation methods and/orthe fragmentation energies varied for the same precursor ion species.This technique allows potential identification of so-called “goldenpairs” of fragments where the different fragmentation techniques producedifferent cleavage mechanisms which are more or less understood. Forexample, a B2 fragment produced by collisionally inducted dissociation(CID) may match a corresponding C2 fragment in ETD, with a fixed massdifference of 17.0265 being the mass of ammonia (NH3).

The method of the present invention can be applied to the analysis ofpolymers and biopolymers such as proteins, peptides, DNA/RNA, lipids andmodifications of these.

1. A method of conducting mass spectrometric analysis, comprising stepsof: sequentially isolating ions of a first and a second precursor ionspecies, the first and second precursor ion species having differentmass-to-charge ratios; combining and storing the isolated ions of thefirst and second precursor ion species; fragmenting the combined ions toyield product ions; and mass analyzing the product ions.
 2. The methodof claim 1, wherein the step of sequentially isolating ions of a firstand second precursor species is performed by an ion trap.
 3. The methodof claim 2, wherein the ions of the first precursor ion species areejected from the ion trap prior to isolating ions of the secondprecursor ion species.
 4. The method of claim 1, wherein the step ofmass analyzing the ions is performed by an Orbitrap mass analyzer. 5.The method of claim 1, wherein the step of mass analyzing the ions isperformed by a time-of-flight (TOF) mass analyzer.
 6. The method ofclaim 1, wherein the step of fragmenting the combined ions is performedat a collision cell.
 7. The method of claim 1, wherein the first andsecond precursor ion species are selected from information contained ina previously acquired precursor mass spectrum.